David A. Wagner

ANALYSIS OF NITRATE, NITRITE AND 15N NITRATE IN BIOLOGICAL FLUIDS

Abstract A new automated system for the analysis of nitrate via reduction with a high-pressure cadmium column is described. Samples of urine, saliva, deproteinized plasma, gastric juice, and milk can be analyzed for nitrate, nitrite, or both with a lower limit of detection of 1.0 nmol NO 3 − or NO 2 − /ml. The system allows quantitative reduction of nitrate and automatically eliminates interference from other compounds normally present in urine and other biological fluids. Analysis rate is 30 samples per hour, with preparation for most samples limited to simple dilution with distilled water. The application of gas chromatography/mass spectrometry for the analysis of 15 NO 3 − in urine after derivatization to 15 NO 2 -benzene is also described.

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [γ2(R43Q)] found in human epilepsy

The γ-aminobutyric acid type A (GABAA) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABAA receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABAA γ2-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABAA receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from α1β2γ2 or α1β2γ2(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABAA receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Palmitoleic acid is elevated in fatty liver disease and reflects hepatic lipogenesis

BACKGROUND Biochemical evidence has linked the coordinate control of fatty acid (FA) synthesis with the activity of stearoyl-CoA desaturase-1 (SCD1). The ratio of 16:1n-7 to 16:0 [SCD1₁₆] in plasma triacylglycerol FA has been used as an index to reflect liver SCD1₁₆ activity and has been proposed as a biomarker of FA synthesis, although this use has not been validated by comparison with isotopically measured de novo lipogenesis (DNL(Meas)). OBJECTIVE We investigated plasma lipid 16:1n-7 and FA indexes of elongation and desaturation in relation to lipogenesis. DESIGN In this cross-sectional investigation of metabolism, 24 overweight adults, who were likely to have elevated DNL, consumed D2O for 10 d and had liver fat (LF) measured by magnetic resonance spectroscopy. Very-low-density lipoprotein (VLDL)-triacylglycerols and plasma free FA [nonesterified fatty acids (NEFAs)] were analyzed by using gas chromatography for the FA composition (molar percentage) and gas chromatography-mass spectrometry and gas chromatography-combustion isotope ratio mass spectrometry for deuterium enrichment. RESULTS In all subjects, VLDL-triacylglycerol 16:1n-7 was significantly (P < 0.01) related to DNL(Meas) (r = 0.56), liver fat (r = 0.53), and adipose insulin resistance (r = 0.56); similar positive relations were shown with the SCD1₁₆ index, and the pattern in NEFAs echoed that of VLDL-triacylglycerols. Compared with subjects with low LF (3.1 ± 2.7%; n = 11), subjects with high LF (18.4 ± 3.6%; n = 13) exhibited a 45% higher VLDL-triacylglycerol 16:1n-7 molar percentage (P < 0.01), 16% of subjects had lower 18:2n-6 (P = 0.01), and 27% of subjects had higher DNL as assessed by using a published DNL index (ratio of 16:0 to 18:2n-6; P = 0.03), which was isotopically confirmed by DNL(Meas) (increased 2.5-fold; P < 0.01). Compared with 16:0 in the diet, the low amount of dietary 16:1n-7 in VLDL-triacylglycerols corresponded to a stronger signal of elevated DNL. CONCLUSION The current data provide support for the use of the VLDL-triacylglycerol 16:1n-7 molar percentage as a biomarker for elevated liver fat when isotope use is not feasible; however, larger-scale confirmatory studies are needed.

A kinetic study of leucine metabolism in severely burned patients. Comparison between a conventional and branched-chain amino acid-enriched nutritional therapy.

A cross-over design study was used to examine the metabolic consequences of enterai feeding for 48 to 96 hours with cither a branched-chain amino acid (BCAA)-enrichcd (44% BCAA) or a conventional egg protein formulation in 12 severely burned adult patients. A stable isotope labeled leucine (L-I-13C-Ieu-cine) tracer approach was used to measure leucine flux and oxidation and to estimate rates of whole body protein synthesis and breakdown. Additionally, 15N2-urca and 6,6-2H-glucose were administered to assess the status of urea and glucose kinetics with these two nutritional treatments. Average patient age was 54 years, and average burn surface area was 36%. Studies were conducted at an average of 25 days postburn. Leucine flux and oxidation were significantly (p < 0.01, by paired t-test) elevated with BCAA feeding as compared to the egg protein formulation. However, there were no significant differences in the rates of leucine incorporation into, or release from, proteins (p > 0.05) between the two dietary periods. Mean rates of body protein synthesis and breakdown for each diet were about twice the rates reported for healthy young adults. Apparent nitrogen balance measurements were not statistically different (p > 0.1) between the two diet periods. Furthermore, urea and glucose kinetics failed to show significant differences between the two diet periods. It appears from these results that the major consequences of increased intake of leucine from the BCAA formula is an enhanced rate of leucine oxidation. In conclusion, (1) the availability of BCAAs is not rate-limiting for enhanced protein synthesis in burn patients, and (2) the use of enriched BCAA formulas in burn therapy does not appear to offer advantages over a routinely used cn-tcral egg protein formula, at least based on the present determinations.

Plasma proline kinetics and the regulation of proline synthesis in man

A quantitative exploration of the regulation of plasma proline concentration, proline oxidation, and proline endogenous biosynthesis was undertaken utilizing a 360-minute primed continuous infusion of L-[1-13C]proline and L-[methyl-2H3]leucine in healthy, postabsorptive young men. The response of proline metabolism to the intravenous administration of two physiologic rates of L-proline, as well as the withdrawal of an L-proline infusion, were examined. The administration of L-proline at 20 mumol.kg-1.h-1 after an overnight fast resulted in a higher steady state plasma proline concentration, attained within 100 minutes, and this was associated with an increase in proline oxidation, from a baseline value of 10.9 to 16.1 mumol.kg-1.h-1 (P less than .01). Additionally, there was a decrease in proline endogenous synthesis from 15.8 (baseline) to 5.3 mumol.kg-1.h-1 (P less than .01). Administration of L-proline at 40 mumol.kg-1.h-1 after an overnight fast resulted again in a higher plasma steady state proline concentration, attained within 100 minutes and with an associated increase in proline oxidation from 13.1 to 20.0 mumol.kg-1.h-1 (P less than .01) and with a decrease in proline endogenous synthesis from 12.2 to -0.6 mumol.kg-1.h-1 (P less than 0.01). The withdrawal of L-proline after a 20 mumol.kg-1.h-1 infusion resulted in a lower plasma steady state proline level and this was accompanied by a decrease in proline oxidation from 21.2 to 18.2 mumol.kg-1.h-1 (P less than .05) and an increase in endogenous synthesis from 22.2 to 29.7 mumol.kg-1.h-1 (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and Spontaneous Open Probability Conferred by the Subunit of the GABAA Receptor

GABAA receptors mediate synaptic and extrasynaptic inhibition. Native receptors consist of α and β subunits, which are required for function, and another “modulatory” subunit, for example, γ, δ, or ϵ. Of these, the ϵ subunit has the most restricted distribution, confers resistance to neurosteroid and anesthetic modulation, and causes spontaneous channel opening. Little is known, however, about how ϵ affects receptor kinetics, which in turn shape responses to both ambient and synaptic GABA exposure. Here, we expressed human α2β1, α2β1γ2, or α2β1ϵ subunit combinations in human embryonic kidney 293 cells and used rapid solution exchange to study receptor kinetics in outside-out patches. The ϵ subunit greatly slowed deactivation and recovery after brief GABA pulses. During long, saturating GABA pulses, the rate of desensitization was slower for α2β1ϵ and α2β1γ2 than for α2β1. However, in α2β1ϵ, the final extent of desensitization was large compared with that of α2β1γ2. Responses in α2β1ϵ, but not the others, were often followed by an “overshoot” above the baseline, suggesting that a fraction of channels are spontaneously open and are transiently silenced by receptor activation and subsequent desensitization. The baseline current and associated noise were reduced by picrotoxin, revealing that ϵ-containing channels are open ∼4% of the time in the absence of GABA. These results suggest that, if ϵ-containing receptors are expressed at synapses, the synaptic currents would be long-lasting but may rundown quickly under high-frequency activation. In addition, silencing of spontaneous openings by desensitization raises the possibility that tonic inhibition mediated by ϵ-containing receptors may be regulated by phasic inhibition.

An Epilepsy-Related Region in the GABA A Receptor Mediates Long-Distance Effects on GABA and Benzodiazepine Binding Sites

The GABAA receptor mutation γ2R43Q causes absence epilepsy in humans. Homology modeling suggests that γ2Arg43, γ2Glu178, and β2Arg117 participate in a salt-bridge network linking the γ2 and β2 subunits. Here we show that several mutations at these locations exert similar long-distance effects on other intersubunit interfaces involved in GABA and benzodiazepine binding. These mutations alter GABA-evoked receptor kinetics by slowing deactivation, enhancing desensitization, or both. Kinetic modeling and nonstationary noise analysis for γ2R43Q reveal that these effects are due to slowed GABA unbinding and slowed recovery from desensitization. Both γ2R43Q and β2R117K also speed diazepam dissociation from the receptor’s benzodiazepine binding interface, as assayed by the rate of decay of diazepam-induced potentiation of GABA-evoked currents. These data demonstrate that γ2Arg43 and β2Arg117 similarly regulate the stability of both the GABA and benzodiazepine binding sites at the distant β/α and α/γ intersubunit interfaces, respectively. A simple explanation for these results is that γ2Arg43 and β2Arg117 participate in interactions between the γ2 and β2 subunits, disruptions of which alter the neighboring intersubunit binding sites in a similar fashion. In addition, γ2Arg43 and γ2Glu178 regulate desensitization, probably mediated within the transmembrane domains near the pore. Therefore, mutations at the γ/β intersubunit interface have specific long-distance effects that are propagated widely throughout the GABAA receptor protein.

A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAA Receptor

The GABAA receptor is a multisubunit protein that transduces the binding of a neurotransmitter at an intersubunit interface into the opening of a central ion channel. The structural components that mediate the steps involved in this action are poorly defined. A large amount of work has focused on clarifying the specific functions and interactions of residues believed to surround the GABA binding pocket. Here, we explored two charged residues (β2Asp163 and α1Arg120), which have been suggested by homology models to participate in a salt-bridge interaction. When mutated to alanine, both single mutants, as well as the double mutant, increase EC50-GABA, decrease the GABA binding rate, and accelerate deactivation and GABA unbinding rates. Double-mutant cycle analysis demonstrates that the effects of each alanine mutation on the GABA binding rate were additive and independent. In contrast, a significant coupling energy was found during an analysis of deactivation time constants. Using kinetic modeling, we further demonstrated that the GABA unbinding rates, in particular, are strongly coupled. These data suggest that β2Asp163 and α1Arg120 form a state-dependent salt bridge, interacting when GABA is bound to the receptor but not when the receptor is in the unbound state.

Whole Body Protein Turnover and Net Protein Balance After Pediatric Thoracic Surgery: A Noninvasive Single-Dose 15N Glycine Stable Isotope Protocol With End-Product Enrichment

BACKGROUND We used the 15 N glycine urinary end-product enrichment technique to quantify whole body protein turnover following thoracic surgery. MATERIALS AND METHODS A single dose of 15 N glycine (2 mg/kg) was administered orally on postoperative day 1 to children (1-18 years) following thoracic surgery. 15 N enrichment of ammonia and urea was measured in mixed urine after 12 and 24 hours, respectively, and protein synthesis, breakdown, and net balance determined. Nitrogen balance (dietary intake minus urinary excretion) was calculated. Urinary 3-methylhistidine:creatinine ratio was measured as a marker of skeletal muscle protein breakdown. RESULTS We enrolled 19 subjects-median (interquartile range): age, 13.8 years (12.2-15.1); weight, 49.2 kg (38.4-60.8)-who underwent thoracotomy (n = 12) or thoracoscopic (n = 7) surgery. Protein synthesis and breakdown by 15 N enrichment were 7.1 (5.5-9) and 7.1 (5.6-9) g·kg-1 ·d-1 with ammonia (12 hours) as the end product, and 5.8 (3.8-6.7) and 6.7 (4.5-7.6) with urea (24 hours), respectively. Net protein balance by the 15 N glycine and urinary urea nitrogen methods were -0.34 (-0.47, -0.3) and -0.48 (-0.65, -0.28) g·kg-1 ·d-1 , respectively (rs = 0.828, P < .001). Postoperative change in 3-methylhistidine:creatinine ratio did not correlate significantly with protein breakdown or balance. CONCLUSION The single-dose oral administration of 15 N glycine stable isotope with measurement of urinary end-product enrichment is a feasible and noninvasive method to investigate whole body protein turnover in children. After major surgery, children manifest increased protein turnover and net negative balance due to increased protein breakdown.

A tight coupling between β2Y97 and β2F200 of the GABAA receptor mediates GABA binding: Coupling of β2Y97 and β2F200 mediates GABA binding

J. Neurochem. (2011) 119, 283–293.